Resolution of racemic delta4-3-ketosteroids



United States Patent RESOLUTION OF RACEMIC A -3-KETOSTEROIDS Theodore I. Feel], King of Prussia, George Greenspan, Narberth, and Leland L. Smith, Malvern, Pa., assignors to American Home Products Corporation, New York, N.Y., a corporation of Delaware No Drawing. Filed Oct. 23, 1963, Scr. No. 318,180

Claims. (6]. 19551) This invention relates to a method of producing steroids having natural configuration and more particularly relates to a method of resolving racemic steroids by microbiological means.

According to the method of the present invention, it has been found that selected racemic steroids of the A4-3- ketone type on treatment with a particular microbiological agent can be resolved in a manner that results in the production of specific steroids having d- (natural) and l- (unnatural) configurations. As used herein, natural configuration means that the steroid molecule will rotate a beam of polarized light to the right or left in a manner similar to the rotational characteristics of the analogous naturally occurring steroids, as determined by conventional test procedures. The present invention therefore encompasses both a new method of preparing particular classes of steroids as well as a method of obtaining steroids of selected optical configuration from sources heretofore unavailable.

More particularly, according to the present invention, it has been found that microbiological dehydrogenation carried out on racemic 13/3-alkyl-l7-substituted-gon-4- ene-3-ones results in the simultaneous production of both 175 hydroxy and 17 keto-13B-alkyl-gona-1,3,5(l0)- trienes having natural configuration. These products may then be separated by chromatography or other means for use as biologically active steroids in themselves or as intermediates in the production of other desired steroids as is described in detail below.

The microbiological dehydrogenation essential to the resolution and production of these natural configuration (d) steroids is accomplished by the action of a microorganism selected from the group consisting of Septomyxa afiinis and Fusarium solani. It has been found that the foregoing fungi are unexpectedly effective in accomplishing the resolution according to the method of the present invention which is illustratively shown by the following reaction sequence:

R R A I.....

Microbiological Dcliydrogenation cal rotatory power as a consequence of their d-configuration, thus corresponding to natural steroids. In terms of quantity of product obtained, generally speaking, more of the steroid corresponding to Formula II in which R and R taken togther are =0 (keto) than when R is OH and R is H. This will be evident from the examples which follow.

It can be seen thusly that the method of the present invention provides a substantially complete resolution of the racemic solution of steroids represented by I into valuable resolved products. The racemic steroid starting materials (I) are obtained by the method described in detail in copending application Serial Number 228,384, filed October 4, 1962, and Belgian Patents 608,370 and 608,369 and the patents cited therein.

In order to effect conversion of the selected racemic steroids of the type encompassed within those defined by Formula I above, it is necessary that the racemic mixture be exposed to the action of a microorganism of the type described. The desired growth of the selected Septomyxa afifnis (NRRL 2746) or Fusarium solani (NRRL 3078) is accomplished in a suitable nutrient medium containing carbohydrate, organic nitrogen and inorganic salts in accordance with well-known technique. The racemic steroid starting material is then dissolved or suspended in a solvent which is nontoxic toward the organism, and added to the cultivated microorganism in a broth medium. This culture is then shaken, aerated or simul taneously aerated and agitated, in order to enhance the growth of the selected fungus and the biochemical conversion of the steroid substrate. The steroid may be added to the broth medium which is then inoculated with the fungus, or the cultivated microorganism in broth medium may be added to the steroid. In certain cases, depending on the conditions of the reaction medium, it may be more desirable to obtain optimum growth of the microorganism before the addition of the steroid.

A useful method for carrying out the process of the present invention is the cultivation of the selected fungus on a suitable nutrient medium under aerobic conditions. A suitable volume of the cell suspension is then seeded into nutrient media of the same or altered composition for supporting growth of the microorganism. The nutrient medium employed may be a yeast extract-dextrose medium, casein hydrolysate, corn steep liquor, water extract of soybean meal or lactalbumin hydrolysate together with an appropriate carbon source.

For example, the selected microorganism can be grown on a dextrose, peptone, corn steep liquor, salts medium at a temperature of about 28 C. for a period of about 64 hours. Suitable nutrient media which may also be used include a yeast extract-dextrose medium or a malt extract-yeast extract-dextrose medium. Other suitable media which contain the necessary carbon, nitrogen and mineral elements in the form of carbon include sugars such as glucose, sucrose, maltose, Xylose, galactose and so forth; alcohols such as glycerol or mannitol; organic acids such as citric acid, maleic acid, acetic acid and various other natural products containing carbohydrates such as corn starch, corn steep liquor, soya bean meal, cotton seed meal and many available materials which have been described heretofore as a source of carbon in steroid fermentation processes. Usually a variety of carbon sources can be employed in the medium with good results. Suitable sources of nitrogen include a number of the above named materials such as corn steep liquor, soya bean meal, cotton seed meal and various other substances such as beef extract, casein, yeast, enzymatically digested proteins, degradation products including peptones, amino acids and many other available proteinaceous materials which have been found to be suitable in supporting the growth of fungi. Inorganic sources of nitrogen include ammonium salts and nitrates which may also be used in the medium as a source of nitrogen to provide a favorable growth medium for the organism.

The mineral requirements of fermentation are usually supplied in the crude materials which are often used as sources of carbon and nitrogen or in the water that is used in the process. However, it may be advisable to supplement the minerals normally present with added amounts to obtain maximum growth. Cations and anions which may be desirable in added amounts include sodium, potassium, calcium, magnesium, phosphate, sulfate, cobalt, manganese and various others. The use of elements such as boron, copper, cobalt, molybdenum and chrominum is often desirable.

The growth of the organism take place under aerobic conditions; and aeration in flasks, for example, can be achieved by agitation on a reciprocating or rotary shaker or in bottles or tanks by forcing sterile air through the fermentation mixture. It is desirable that the sterile air be forced through the medium in an amount of from /2 to 2 volumes of air per volume of medium per minute. Agitation in the bottles or fermenter tanks is provided by mechanical impeller. It is preferable to carry out the process of the present invention at a temperature within the range of about 15 C. to about 37 C.

After the growth of the microorganism has taken place to some extent, the steroids are added to the fermentation broth in solution or finely divided form. One of the preferred methods is to dissolve the steroid in methanol or other water-miscible solvents and add it to the fermentation medium at the desired stage in the process. Although the steroid may precipitate from solution when so added, it is disposed through the medium as a fine suspension and becomes readily available to the organism for oxidation. The amount of steroid added to the fermentation medium may vary considerably but is generally about to 1 gram per liter of medium.

Recovery of the desired product is accomplished by extraction with a suitable water immiscible solvent followed by filtration, adsorption or other of the commonly used procedures practiced in the art of steroid recovery under similar conditions. The purified products may be obtained following extraction of fermentation mixture by chromatography, fractional crystallization, counter-current distribution or other conventional steroid separation techniques. When extraction is used to recover the steroid product, chlorinated lower hydrocarbons, ketones, esters or alcohols may be used. Included within these solvents would be chloroform, methylene chloride, trichloroethane, ethylene dichloride, butanol, diethylketone, methyl isobutyl ketone, ethyl acetate and the like.

The resolved products of the present invention and more specifically the d-steroids encompassed within Formula II above are valuable for their estrogenic activity. These resolved steroids are also useful as intermediates in the synthesis of the l9-nor-A -3-ketosteroids, which are useful as androgenic hormones. The following reaction sequence is illustrative of the manner in which the resolved steroids, obtained according to the method of the present invention, may be utilized as intermeidates in the preparation of other valuable steroids, in particular those having valuable androgenic, anabolic and progestational properties such as the products VI below:

R1 R2 1..... p

(CH3)2SO4 HO alkali crno- /Li/NH 1! In the foregoing sequence, steroid II is a selected resolved product of the present invention in which R R and R have the values previously ascribed. The intermediate steroids III, IV and V are obtained as suggested by the reaction identified and lead to the desired products VI, which are useful for their androgenic, anabolic and progestational properties.

Reference now to the specific examples which follow will provide a better understadning tof the method of the present invention and to the resolved steroid products which are obtainable thereby.

Example I An agar slant of Septomyxa ajfim's NRRL 2746 is washed with 5 ml. of distilled water and the resulting suspension transferred to a 250 ml. flask containing 50 ml. of the following medium: yeast extract 1%, dextrose 1% and distilled water ml.

The flask is incubated on a reciprocating shaker 83 strokes/min, 4" strokes, 28 C., for 96 hours, at which time, a 10% transfer is made to another flask of the same medium. After 30 hours of shaking, dl-l3fl-ethyl-l7fihydroXygon-4'en-3-one is added in MeOH to give a concentration of 0.25 g./l. After 20 hours of further incubation on a rotary shaker, a 5 ml. sample is taken and extracted with 1 ml. of methyl isobutyl ketone. The extract is spotted on No. 4 Whatman paper and the papergram run in a toluenepropylene glycol system. After drying, the chromatogram is sprayed with a mixture of equal parts of 1% solutions of K Fe(CN) and FeCl;.;. Two products positive to the Turnbull blue stain appear on paper, identified as 13fl-ethyl-3-hydroxygona-1,3,5(10)- trien 17 one and l3,8ethylgona-1,3,5(10)fitriene-3,17 8- diol.

Example 11 Eight agar slants of Septomyxa afiinis NRRL 2746 are each washed with 5 ml. of distilled water. One half the volume of each suspension is transferred to sixteen 250 ml. flasks, each containing 50 ml. of the medium described in Example I. The flasks are incubated on a reciprocating shaker, 28 C., for 66 hours. The contents of single flasks are then transferred to sixteen 2 liter flasks, each with 400 ml. of the same medium and the flasks shaken on the reciprocating shaker for 29 hours. all 13B ethyl 17,8 hydroxygon 4 en 3 one, dissolved in methanol, is added to give a concentration of 0.35 g./l. The flasks are further incubated on a rotary shaker, 250 r.p.m., 28 C.

The course of the transformation is followed by paper chromatography and the flasks harvested at 89.5 hours. The total steroid fermented is 2.06 g.

Fermentation broth obtained by the action of Septomyxa afiinis NRRL 2746 on 2.06 g. of race-mic 13[3 ethyl-17B-hydroxygon-4-en-3-one is extracted three times with equal volumes of ethyl acetate, and the ethyl acetate extracts are combined. The solvent extract is concentrated under vacuum to a volume of ten milliliters and then chilled. The precipitated solids are filtered, washed with methanol, and dried, yielding 325 mg. of product which is shown to be almost pure 13fl-ethyl-3-hydroxygona 1,3,5 trien 17 one by thin-layer chromatographic analysis. The product is recrystallized from boiling methanol and a first crop of 196 mg. of pure product, [a] +133.7 (0.5% in ethyl acetate), ti -13B- ethyl-13-hydroxygona-1,3,5(10)-trien-17-one is recovered.

Example III Following a procedure similar to that set out above, racemic 17/9 hydroxy 13,8 propylgon 4 ene 3- one is brought into contact with Septomyxa afiinis to produce d 13;? propylgona 1,3,5(10) triene 3,176- diol and d 3 hydroxy 13(3 propylgona 1,3,5(10)- trien-17-one.

Example IV According to the method described above, d-13fl-butyl- 3 hydroxygona 1,3,5( 10) trien 17 one and d- 135 butylgona 1,3,5(10) triene 3,175 diol are prepared by contacting dl-"l3,8-butyl-17fl-hydroxygon-4-ene- 3-one with Septomyxa afiinis.

Example V An agar slant of Fusarium solani (NRRL 3078) is washed with 5 ml. of distilled water and half the result ing suspension transferred to a 250 ml. flask containing 50 ml. of the following medium yeast extract 1%, dextrose 1% and distilled water 100 ml.

The flask is incubated on a rotary shaker 250 rpm. 28 C., for 69 hours, after which a 10% mycelial transfer is made to a flask of the same medium. Following 24 hours of further shaking, dl-l3fl-ethyl-17B-hydroxygon- 4-ene-3-one is added in methanol to give a concentration of 0.25 g./l.

Five ml. samples are taken at 1, 2, 3 and 4 days and each sample extracted with 1 ml. of methyl isobutyl ketone. The extracts are spotted on No. 4 Whatman paper and the papergram run in the toluene-propylene glycol system.

After drying, the chromatogram is sprayed with a mixture of equal parts of 1% solutions of K Fe(CN) and FeCl Two products positive to Turnbull blue stain appear on the paper, namely 13p-ethyl-3-hydroxygona- 1,3,5(10) trien 17 one and 13,8 ethylgona 1,3, 5(10)-triene-3,17fi-diol.

Example VI Five agar slants of Fusarium solani (NRRL 3078) are washed with 5 ml. of distilled water and one third of each of the resulting suspensions transferred to fourteen 250 ml. flasks containing 50 ml. of the medium utilized in Example V. Incubation of the flasks 0n the rotary shaker at 28 C. for 69 hours is followed by 9% mycelial transfers to each of sixteen 2 liter flasks with 400 ml. of the same medium. The flasks are incubated as above for 24 hours.

dl 13B ethyl 17,8-hydroxygon 4 en 3 one, dissolved in methanol, is added to give a concentration of 0.35 g./l. The flakes are incubated as above and the transformation followed by paper chromatography. The flasks are harvested for extraction at 77 hours; a total of 2.219 g. is fermented.

Fermentation broth obtained by the action of F usarz'um solani on 2.219 g. of dl-13,8-ethyl-17fl-hydroxygen-4-en- 3-one is extracted with ethyl acetate after filtration of the mycelium. The ethyl acetate extracts are evaporated to ca. 25 ml. and chilled overnight. The precipitated material weighs 648 mg. and is analyzed by thin-layer chromatography to be a mixture of unaltered substrate, major product (recognized as 13B-ethyl-3-hydroxygona-1,3, 5(10)-trien-17-one) and minor product (recognized as 13,8 ethylgona 1,3,5(10) triene 3,17B-diol). The product is dissolved in boiling chloroform-methanol (1:1), cooled to room temperature, and then chilled for one hour. The precipitated product weighs 666 mg. and analyzes as pure d-l3fi-ethyl-3-hydroxygona-1,3,5(10)- trien-17-one, M.P. 249-252 C.; [o] (0.5% in ethyl acetate).

By careful column chromatography of .the mother liquor from the above example, the remaining unaltered substrate and the minor product d-l3;8-ethylgona-l,3,5(10)- triene-3,17B-diol can be isolated in pure form.

Example VII The procedure of Example VI is repeated using the following medium: dextrose 50 g., peptone 20 g., corn steep liquor 5 g., and distilled water 1:1. Two products positive to Turnbull blue are noted as in Example V.

While the compounds of the present invention and the manner in which they are prepared have been described with some degree of particularity, it is to be understood that the invention is not so limited. The invention is .to be limited only to the extent required by law in accordance with the claims appended hereto.

The invention claimed is:

1. The method of preparing compounds of the structure:

wherein R is lower alkyl having more than one carbon atom; R is OH and R is selected from the group consisting of H, lower alkyl, lower alkenyl and lower alkynyl and R and R taken together are =0, which comprises treating a compound of the structure:

wherein R R and R represent the substituents above described with a fungus selected from the group consisting of Fusarium solani and Septomyxa afiinis and recovering and separating the desired compounds.

2. The method as claimed in claim 1 wherein the fungus is F usarium solzmi.

3. The method as claimed in claim 1 wherein the fungus is Septomyxa afiinis.

4. The method as claimed in claim 1 wherein the starting compound is 13 8-a1kyl-17B-hydroxygon-4-en-3-one.

5. The method as claimed in claim 4 wherein the starting compound is 13(3-ethyl-17fi-hydroxygon-4-en-3-one.

6. The method as claimed in claim 4 wherein the starting compound is 13,43-propyl-17/3-hydroxygon-4-en-3-one.

7. The method as claimed in claim 1 wherein the desired compounds are separated by chromatography.

8. The method of resolving racemic steroids having the structure:

wherein R is lower alkyl having more than one carbon atom; R is hydroxy; and R is selected from the group consisting of hydrogen, lower alkyl, lower alkenyl, lower alkynyl which comprises dehydrogenating a racemic steroid of structure I with a microorganism selected from the group consisting of Fusarium solani and Septomyxa afi'inis,

(A)- to produce a compound of the structure:

References Cited by the Examiner UNITED STATES PATENTS 2,897,218 7/1959 Sebek et a1 260397.45 3,022,226 2/1962 Ross 195-51 3,102,896 9/1963 Babcock et al. 260-397.45

A. LOUIS MONACELL, Primary Examiner.

D. M. STEPHENS, Assistant Examiner. 

1. THE METHOD OF PREPARING COMPOUNDS OF THE STRUCTURE: 